CAJ | 학술논문

目的在对肠病毒71型(enterovirus 71,EV71)分离株进行鉴定的基础上,对其诱导小鼠产生的免疫应答水平进行研究,拟为进一步的EV71候选疫苗研究奠定基础.方法超速离心纯化病毒后,采用磷钨酸负染法通过电镜观察病毒形态、大小;采用特异性EV71单抗通过间接免疫荧光法(IFA)检测病毒的特异性;合成EV71特异性引物,通过RT-PCR对病毒进行分子生物学鉴定;病毒的VP1基因序列测定后,与其他EV71序列进行比较,绘制种系发育树,确定病毒基因型;通过腹腔注射途径免疫小鼠,免疫后分离血清通过微量细胞病变抑制法测定血清中和抗体效价,ELISA法检测血清抗体水平和抗体亚型水平.结果电镜下可观察到典型的圆形病毒颗粒,直径为20～30 nm,呈典型的肠病毒形态;085和087株病毒感染细胞中均能观察到黄绿色荧光,提示该两株病毒可与EV71单抗特异性结合;RT-PCR可以从病毒感染细胞中扩增出226 bp大小的特异性产物带,而采用CA16特异性引物则不能扩增出相应大小的产物带;基于VP1基因序列的种系发育分析显示,087和085株均为C4基因型;085和087株EV71免疫小鼠后可诱导产生能中和包括其本身在内的多个病毒株的中和抗体,针对同一株中和病毒,实验组间差异无统计学意义;针对不同中和病毒株,各实验组中和本株、523-07T株的能力明显高于FY-02T株(P<0.05);ELISA法检测结果显示,接种灭活前后的EV71病毒085和087株后均能诱导小鼠产生抗EV71特异性IgG;IgG亚型为IgG1/IgG2a混合型,灭活病毒免疫组小鼠血清EV71特异性IgG、IgG1、IgG2a水平明显高于活病毒免疫组(P<0.05).两株病毒之间差异无统计学意义.结论本研究分离到的085和087株病毒均为EV71 C4基因亚型,并具有一定的免疫原性.
목적 재대장병독71형(enterovirus 71,EV71)분리주진행감정적기출상,대기유도소서산생적면역응답수평진행연구,의위진일보적EV71후선역묘연구전정기출.방법 초속리심순화병독후,채용린오산부염법통과전경관찰병독형태、대소;채용특이성EV71단항통과간접면역형광법(IFA)검측병독적특이성;합성EV71특이성인물,통과RT-PCR대병독진행분자생물학감정;병독적VP1기인서렬측정후,여기타EV71서렬진행비교,회제충계발육수,학정병독기인형;통과복강주사도경면역소서,면역후분리혈청통과미량세포병변억제법측정혈청중화항체효개,ELISA법검측혈청항체수평화항체아형수평.결과 전경하가관찰도전형적원형병독과립,직경위20～30 nm,정전형적장병독형태;085화087주병독감염세포중균능관찰도황록색형광,제시해량주병독가여EV71단항특이성결합;RT-PCR가이종병독감염세포중확증출226 bp대소적특이성산물대,이채용CA16특이성인물칙불능확증출상응대소적산물대;기우VP1기인서렬적충계발육분석현시,087화085주균위C4기인형;085화087주EV71면역소서후가유도산생능중화포괄기본신재내적다개병독주적중화항체,침대동일주중화병독,실험조간차이무통계학의의;침대불동중화병독주,각실험조중화본주、523-07T주적능력명현고우FY-02T주(P<0.05);ELISA법검측결과현시,접충멸활전후적EV71병독085화087주후균능유도소서산생항EV71특이성IgG;IgG아형위IgG1/IgG2a혼합형,멸활병독면역조소서혈청EV71특이성IgG、IgG1、IgG2a수평명현고우활병독면역조(P<0.05).량주병독지간차이무통계학의의.결론 본연구분리도적085화087주병독균위EV71 C4기인아형,병구유일정적면역원성.
Objective To identify two enterovirus 71 (EV71) isolates and evaluate their immunogenicity for the purpose of EV71 vaccine development.Methods The morphology of EV71 isolates was determined by electronic microscopy,both of the 087 and 085 isolates were identified by indirect fluorescence assay (IFA) using anti-EV71 monoclonal antibody and by RT-PCR with EV71 specific primers,the genotype was determined by phylogenetic analysis based on VP1 gene sequence.The immunogenicity was evaluated on BALB/c mice by the detection of neutralization antibody and anti-EV71 IgG in the serum with neutralization test and ELISA.Results The round virus particles with 20-30 nm in diameter can be objected by electronic microscopy in the virus infected cells culture which is typical for enterovirus.Both 087 and 085 strains can react with anti-EV71 monoclonal antibody,the positive virus infected cells stained with FITC can he detected by IFA.The 226 bp products were amplified from the total RNA extracted from virus infected cells by RT-PCR with EV71 special primers,not CA16 special primers.The genotype of both strains is CA,which is identical to the other epidemiological EV71 strains in mainland,China in the latest decade.Both strains can induced high level of neutralization antibody before or after inactivation which can neutralize more EV71 strains than themselves,for the same neutralized virus strain,no difference was found between 085 and 087 strains and between live and inactivated group for each strain either,but the ability to neutralize FY-02T strain for the serum from all the immunized groups is lower than other strains significantly(P<0.05).Compared with control,anti-EV71 IgG can be detected in the sera from immunized mice with the subtype IgG1/IgG2a by ELISA,which are not different between 087 and 085 strains but higher in inactivated virus group.Conclusion Both of the two EV71 isolates,085 and 087 strains were identified to be EV71 with genotype C4 and are highly immunogenic.